Our invention relates to cells consisting of hemoglobin encapsulated in lipids and more especially phospholipids and to the method of making such synthetic cells. These cells are characterized by comparable O.sub.2 --CO.sub.2 conjugation and transference to that of naturally occurring red blood cells. Furthermore, our synthetic cells are of such small size and flexibility to readily pass through mammalian capillary systems where such O.sub.2 --CO.sub.2 transfer takes place. Another, most desirable feature of our cells is that their use introduces no foreign matter to the recipient.
Our synthetic cells, in terms of oxygen carrying capability, function very similarly to normal mammalian red blood cells and accordingly in suspension offer substantial utility as a transfusion liquid. Such cells appear as acceptable to the mammalian host as are natural such cells, function in substantially the same manner and should be metabolized and excreted as are naturally occurring cells.
As is known to those skilled in this art, hemoglobin is a conjugated protein having a prosthetic group -- heme -- affixed to the protein, globin. It is the red coloring matter of blood and is found, contained, in the red blood cells. Its essential utility stems from its ability to unite in loose combination with atmospheric oxygen to form oxyhemoglobin. In mammals this occurs in the capillaries adjacent the lung alveoli to produce so-called oxygenated blood. This is carried in the arterial system to the tissues where a portion of the oxygen is released and then the venous blood, partially depleted in oxygen, is returned to the lungs for further oxygenation.
As further background we note that heme is an iron porphyrin, i.e., the union of iron with four pyrrole groups. The iron is basically in the ferrous state. Hemoglobin is usually designated as ##STR1##
Thus hemoglobin is a tetramer consisting of four sub-units; each sub-unit is a combination of a polypeptide chain, which is the protein or globin part of hemoglobin, and a heme. The latter is the functional unit or active site to which oxygen may be bound.
Whole blood, especially human, when drawn for transfusion purposes, is considered to have a storage life of 21 days. By present regulation, such blood 21 days old must be discarded and no longer used for blood transfusion. As a practical matter upon the passage of such time, the red cells break down thus making the old blood substantially useless for its intended purpose. However, such "old blood" still contains useful, functional hemoglobin and can be used as the starting material in the preparation of the present cells.
In distinction to the aging problem -- 21 days -- encountered with whole, natural blood, we find that the present cells when appropriately buffered, have quite an extended, useful, shelf life, as is noted below.
Our synthetic hemoglobin cells offer another advantage -- because of how they are made they can be considered to be in the class of universal donor. Whole blood for transfusion purposes must be typed and extreme care taken to assure compatability with the blood type of the recipient. This is not the case with the present cells. Our starting material for encapsulation is what is commonly referred to as "stroma-free" hemoglobin. This is hemoglobin free of the cell membranes or membrane fragments of the red blood cells but having associated therewith the normal cell components, such as diphosphoglycerate and carbonic anhydrase, required for O.sub.2 --CO.sub.2 exchange. The membranes of natural erythrocytes contain many different proteins and it is such protein which necessitates blood-typing. The membranes of our synthetic cells are essentially formed of universally present (i.e., in the mammal) lipids and the like which are not subject to antigenic reactions of proteins.
As further background to our invention, we note that the separation of red blood cells from whole blood is an old, established common practice. And the separation of hemoglobin from its associated red cells by a multitude of techniques is similarly well-known. Such two known categories of techniques represent the starting points in forming the hemoglobin cells of this invention.
As further background we also note that some workers have been investigating the use of cell-free hemoglobin solutions as natural blood substitutes. These solutions suffer the disadvantage of being rapidly excreted by the body and thus really do not accomplish their intended purpose for anything but the shortest of times. In distinction to this the present lipid encapsulated hemoglobin cells will be retained by the body for extended and more useful periods.
We also note that prior workers have formed what are generically referred to as liposomes.
Among the prior patents we note the following:
Bower, U.S. Pat. No. 2,527,210 is directed to a hemoglobin solution wherein a freezing-thawing technique is used to destroy the red cell membranes.
Childs, U.S. Pat. No. 3,133,881 discloses a centrifugation method which may be employed to separate red cells from the other constituents of whole blood.
Van Dyck et al., U.S. Pat. No. 3,351,432 is directed to the washing and reconstituting of red blood cells and Ushakoff, U.S. Pat. No. 3,418,209 to making red cells storable by a combination with a glycerin solution. Similarly, Ilg, U.S. Pat. No. Re. 27,359 is directed to the washing of red cells.
Bonhard, U.S. Pat. No. 3,864,478 discloses another method of making a hemoglobin solution.
The prior art teachings in no way suggest or hint at the lipid encapsulation of hemoglobin to form the present synthetic cells nor the benefit or utility of such cells. The similarity of the present synthetic cells to normal red blood cells in terms of oxygen carrying capability is unexpected.
Accordingly, a principal object of our invention is to provide lipid encapsulated hemoglobin -- synthetic erythrocytes -- and a method for their manufacture. How this is accomplished is set out as this description proceeds.